Application of 5-hydroxytryptamine (5-HT) to the lamprey spinal cord in vitro reversibly depressed the chemical component of excitatory postsynaptic potentials recorded intracellularly in motoneurons and evoked by stimulation of single reticulospinal Muller cells. The depression could be produced either by local application of small volumes of 10 mM 5-HT to the surface of the spinal cord or by bath-application of 1 or 10-mu-M 5-HT. No effect on the input resistance of the postsynaptic cells or their sensitivity to glutamate, the suspected transmitter at this synapse, could be detected, suggesting the possibility of a presynaptic action of 5-HT at this synapse in the lamprey.